Mannose binding lectin and uses thereof

ABSTRACT

The present inventors have shown that MASP-depleted MBL is able to recruit MASPs from plasma and successfully activate complement cascade. Furthermore, it has been discovered that MBL purified as a complex has limited ability to activate the complement cascade when compared to MASP-depleted MBL. Accordingly, the present invention provides a pharmaceutical composition comprising an isolated non-recombinant mannose binding lectin (MBL) substantially free from activated MBL associated serine proteases (MASPs) together with a pharmaceutically acceptable carrier or diluent. Also provided is a method of treating a subject in need of MBL comprising administering to the subject an effective amount of a pharmaceutical composition of the invention.

FIELD OF THE INVENTION

The present invention relates to purified mannose binding lectin (MBL),substantially free from MBL associated serine proteases (MASPs) and itsuse in therapy.

BACKGROUND TO THE INVENTION

Mannose binding lectin (MBL), sometimes referred to as mannan bindinglectin or mannose binding protein, is a liver derived C-type serumlectin with structural homology to complement component C1q. MBL canactivate complement via the lectin and classical pathways, and caninteract with specific C1q-like receptors on the surface of phagocytes,thus playing an important role in first-line host defence.

MBL is a member of the collectin family of proteins that arecharacterised by the presence of both a collagenous region and aglobular lectin domain. The structural unit of MBL is a 96 kDa collagentriple helix of three 32 kDa subunits, each with acarbohydrate-recognition domain. The helix is stabilised by disulphidebonds between N-terminal cysteines. MBL oligomerizes as multiples ofthis 96 kDa unit and the native protein is commonly found as trimers tohexamers ranging from 270 kDa to approximately 650 kDa. MBL fullfunctionality is only obtained when it is in its higher oligomericforms. There is evidence that MBL must at least be tetrameric to enableeffective complement activation. This oligomeric structure allows MBLmultiple ligand binding sites and mimics the multiple bindingcharacteristics of IgM.

MBL binds many different sugars, but binds most avidly to mannose andN-acetylglucosamine. These sugars are prevalent on the cell walls ofmany pathogens such as yeast, gram negative enteric bacteria, grampositive bacteria, mycobacteria, some viruses, and certain parasites. Asmost of the MBL sugar targets are not expressed at high densities on thesurface of mammalian cells, MBL has the ability to distinguish self fromnon-self. MBL thus serves as a pattern recognition molecule in thefirst-line of host defence, a central part of the so-called innateimmune system (Turner, 1996).

Central in the efficient and effective complement activation function ofMBL is its close association in vivo with at least two pro-enzymescalled MBL associated serine proteases 1, 2 and 3 (MASP1, MASP2 andMASP-3). These single polypeptides of 93 kDa, 76 kDa and 105 kDa,respectively become activated when MBL binds its ligand and promoteefficient complement activation via the lectin pathway (Turner, 1996).It has been demonstrated that MASP2 is essential for complementactivation and this enzyme alone is capable of initiating the complementcascade without the presence of either MASP1, or the recently describedMASP3. MASP2 is thus the critical enzyme associating with MBL to promoteactivation of the complement cascade.

The MBL gene (MBL2) is located on chromosome 10 at 10q11.2-q21 andcontains four exons. A number of mutations in MBL2 that have an impacton the expression of functional protein have been described. Singlenucleotide substitutions in codons 52, 54 and 57 of exon 1 of the MBL2gene are believed to disrupt the assembly of MBL subunits into the basictrimeric structural unit.

In addition, at least two polymorphisms have been described in thepromoter region (at positions −550 and −221 respectively) that alter thelevel of expression of individual MBL sub-units. The frequency ofmutations in the MBL gene varies among ethnic groups. For example thecodon 54 variant occurs with a frequency of 15% in Caucasians while thecodon 57 variant is seen exclusively in Africans. The practicalsignificance of the common occurrence of both the gene mutations and thepromoter polymorphisms is that MBL deficiency is relatively common inthe general population. The serum level of MBL in individuals homozygousfor the wild-type gene ranges from 1 to 5 μg/mL while those individualshomozygous for MBL2 mutations have levels of 5 to 25 ng/mL andheterozygous individuals have levels approximately ⅛^(th) normal, butthere is considerable observed variation in levels.

A number of lines of evidence suggest that MBL deficiency has clinicallyimportant consequences.

A childhood syndrome of recurrent infections, failure to thrive andchronic diarrhoea was first linked to an in vitro opsonic defect ofplasma in 1968. It was subsequently confirmed that this syndrome wasassociated with low MBL levels in 10 children aged from 15 mths to 9yrs. The importance of MBL2 deficiency as a risk factor for childhoodinfection was confirmed in a consecutive series of 345 children admittedto hospital with infection. The prevalence of MBL2 gene mutations inchildren with infection was twice that in those without infection andthe increased susceptibility was seen in both heterozygote andhomozygote individuals. Infections seen ranged from chest infections andotitis media through to life threatening meningococcaemia.

The association of MBL deficiency with meningococcal disease in childrenhas been confirmed in a large study of 266 cases. 7.7% of the hospitalbased cases were homozygous for MBL polymorphisms in comparison to 1.5%of the control group giving an odds ratio of 6.5. It was concluded thatthe genetic variants of MBL may account for a third of all cases.

These data in the paediatric population have led to the hypothesis thatthe major role of MBL is to provide protection during the so called“window of vulnerability” that occurs after maternal antibodies are lostand before the maturation of an infants own antibody repertoire (6 mthsto 18 mths).

The recent findings that MBL genotypic variants are associated with anearly age of onset of presentation of common variable immunodeficiencyand acute lymphoblastic leukaemia adds weight to the hypothesis that MBLmediated host defence takes on greater importance when other componentsof the immune system are immature or impaired.

Common genetic variations in the MBL gene have recently been associatedwith increased disease severity and risk of infection with Burkholderiacepacia in 149 cystic fibrosis (CF) patients. MBL variant alleles werealso associated with poor prognosis and early death—predicted age ofsurvival was reduced by 8 years in variant allele carriers when comparedwith normal homozygotes in the CF population.

There is increasing evidence of the clinical importance of MBLdeficiency in adults. Four adult patients with “severe and unusual”infections (including recurrent skin infections, Cryptosporidiosis,Meningococal meningitis with recurrent herpes simplex and oesophagealcandidiasis, and Klebsiella pneumonia) were shown to have MBL2 mutationsinvolving either codons 52 or 54.

In 228 adult patients suspected of having non-HIV-relatedimmunodeficiency, the frequency of heterozygosity for MBL2 mutations wasthe same as a control population. However, there was a significantincrease in homozygous MBL2 mutations amongst those with presumedimmunodeficiency (8.3% vs 0.8%). Data have also been presented showingthat the risk of HIV infection is greater and the rate of progression ofAIDS is faster in men homozygous for MBL polymorphisms.

In patients in whom the adaptive immune response has been compromised bychemotherapeutic regimens, the effect of MBL structural gene mutationsand low levels of circulating MBL has been clearly associated withincreased incidence of infection and severity of infection. Adultsreceiving chemotherapy for haematological malignancies with MBL levelsbelow 0.5 μg/ml had significantly increased incidence and severity ofinfection. Donor and recipient MBL genotype were found to be importantin influencing the risk of infection in adults following allogeneic stemcell transplantation. Amongst 100 children undergoing chemotherapy,those with structural MBL gene mutations had twice as many days offebrile neutropenia as those with wild type MBL genes and four of thesewere admitted to ICU with infection. MBL levels less than 1 μg/ml werethought to be critical in this study.

In one of the few prospective, community based studies yet performed 252children were examined (Koch et al., 2001). It was discovered that MBLdeficiency as strongly linked (twice the risk) to acute respiratoryinfection in children aged 6 to 17 months. MBL deficiency had lessimpact in those aged 0 to 5 months and had no impact on acuterespiratory infection in those aged 18 to 23 months.

MBL-MASP complex has been purified routinely on a laboratory scale since1980. MBL-MASP complex purification has been performed by affinitychromatography in various forms. The ligand is usually yeast mannan(Anderson et al., 1992; Holmskov et al., 1993). One or two cyclesthrough the column are performed with the first elution with high saltor EDTA (Koppel et al., 1994; Anderson et al., 1992; Holmskov et al.,1993) and the final elution with mannose (Koppel et al., 1994; Andersonet al., 1992; Matsushita et al., 1992; Holmskov et al., 1993).

Human MBL-MASP complex has also been purified from a waste fractionproduced during the fractionation of plasma proteins, on a laboratoryscale (Kilpatrick, 2000), and under GMP conditions at the Statens SerumInstitut (Valdimarsson et al., 1998). Scottish Cohn fraction m is awaste product of IgG production by plasma fractionation. Cryosupernatantproduced from plasma is precipitated with 21% ethanol. The precipitatefrom this step is called fraction I+II+III. A further precipitation with8% ethanol produces fraction I+m from which MBL-MASP complex can beaffinity captured using an Emphaze-mannan column. Elution of MBL-MASPcomplex was achieved with first EDTA, then mannose solutions. The yieldof MBL-MASP complex from this procedure is quoted as 10 mg/kg offraction I+III paste; a specific activity seven fold greater than pooledplasma (Kilpatrick, 2000). In this way, highly pure MBL-MASP complex(300-600 μg/litre plasma) can be recovered with simple mannose elution.

An alternative purification technique is discussed in WO99/64453 whichdiscloses a chromatographic purification step using a non-conjugatedpolysaccharide matrix.

SUMMARY OF THE INVENTION

The present inventors have discovered that MASP-depleted MBLcompositions are superior at activating the complement cascade whencompared to MBL purified in complex with its associated MASPs.Consequently, the present inventors have found that for the purpose offormulating a safe, effective therapeutic product for administration tosubjects, MASPs, or at least activated MASPs should be removed from theMBL during, or prior to, or after purification of the MBL-MASP complex

Accordingly, in a first aspect, the present invention provides apharmaceutical composition comprising isolated non-recombinant mannosebinding lectin (MBL) substantially free from activated MBL associatedserine proteases (MASPs) together with a pharmaceutically acceptablecarrier or diluent.

Preferably the composition is substantially free of MASPs, whetheractivated or not. Typically, the MBL is human MBL.

The present inventors have found that MASP-depleted MBL is able torecruit MASPs from plasma to produce a functional complex that cansuccessfully activate the complement cascade. In contrast, it appearsthat purified MBL-MASP complex has a limited capacity to recruitproenzyme (or fresh) MASP. This is probably due to the presence in thepurified complex of activated MASP attached to the binding sites on MBLas a result of activation during the purification process (e.g. beingactivated upon binding to mannan columns), the activated MASP beingdifficult to displace. By contrast, proenzyme MASPs can be freshlyrecruited to available binding sites on purified MASP-depleted MBL. Thisalso restores the regulation component of the MBL-MASP complex, makingit a safer, more effective therapeutic product.

In a preferred embodiment, the MBL is obtained by a method comprising:

-   (i) providing a complex of non-recombinant MBL and one or more    MASPs;-   (ii) incubating the complex in a suitable buffer to dissociate the    MBL from the one or more MASPs; and-   (iii) separating the MBL from the one or more MASPs.

Preferably, the buffer in step (ii) is an EDTA/acetate buffer at a pH offrom 4.0 to 5.0.

Furthermore, it is preferred that the buffer comprises NaCl. Preferably,the buffer has an NaCl concentration of at least 0.5 M. More preferably,the buffer has an NaCl concentration of about 1 M.

Preferably step (iii) includes a chromatographic method and/orfiltration. In a further preferred embodiment, the chromatographicmethod is selected from the group consisting of: size exclusionchromatography and ion exchange chromatography.

In a second aspect, the present invention also provides a method ofproducing a pharmaceutical composition, the method comprising:

-   (i) providing a complex of non-recombinant MBL and one or more    MASPs;-   (ii) dissociating the MBL from at least some of the one or more    MASPs;-   (iii) separating the MBL from at least some of the one or more    MASPs; and-   (iv) admixing the resulting MBL from step (iii) with a    pharmaceutically acceptable carrier or diluent.

Preferably, step (ii) involves incubating the complex in a suitablebuffer.

Preferably, the buffer is an EDTA/acetate buffer at a pH of from 4.0 to5.0.

Furthermore, it is preferred that the buffer comprises NaCl. Preferably,the buffer has an NaCl concentration of at least 0.5 M. More preferably,the buffer has an NaCl concentration of about 1 M.

Preferably step (iii) includes a chromatographic method and/orfiltration. In a further preferred embodiment, the chromatographicmethod is selected from the group consisting of: size exclusionchromatography and ion exchange chromatography.

In a preferred embodiment step (i) comprises providing a side fractionfrom plasma fraction processes. Preferably step (i) further comprisesseparating complexes of non-recombinant MBL and one or more MASPs fromother plasma proteins present in the side fraction from plasma fractionprocesses by mannan affinity chromatography.

The present invention also provides a pharmaceutical compositionobtained by the method of the second aspect of the invention.Preferably, the composition is substantially free of activated MASPs

In another aspect, the present invention provides a method of treatingor preventing a disease in a subject, the method comprisingadministering to the subject an effective amount of a pharmaceuticalcomposition of the invention.

The disease can be any condition, the treatment or prevention of whichwould be aided by the subject being administered with purifiedMASP-depleted MBL. Examples of suitable recipients of the methodinclude, but are not limited to, bone marrow allograft recipients,subjects with cystic fibrosis, subjects with an immunodeficiency,subjects with acute lymphoblastic leukaemia, subjects with communityacquired or nosocomial septicaemia, subjects with or susceptible to aninfection by a pathogen, low birthweight and/or premature infants.Typically, the subject has an MBL deficiency.

The present invention also provides a composition comprising isolatednon-recombinant MBL, said composition being substantially free of MASPs,for use prophylactically or in therapy.

The present invention further provides the use of a compositioncomprising isolated non-recombinant MBL, said composition beingsubstantially free of MASPs, in the manufacture of a medicament for usein administering to a subject in need of said composition.

Examples of suitable recipients include, but are not limited to, bonemarrow allograft recipients, subjects with cystic fibrosis, subjectswith an immunodeficiency, subjects with acute lymphoblastic leukaemia,subjects with community acquired or nosocomial septicaemia, subjectswith or susceptible to an infection by a pathogen, low birthweightand/or premature infants. Typically, the subject has an MBL deficiency.

The present inventors have also devised cleavage substrates, and assaysfor the use thereof, for determining the levels of MASP activity in asample. Such assays can be used for monitoring MBL purificationprocedures described herein, or for any other purpose where it isdesirable to analyse MASP activity.

Thus, in a further aspect the present invention provides a peptide offormula X-R1-Arg-R2-Y wherein R1-Arg-R2 is a peptide consisting of 6 ormore contiguous amino acids derived from the MASP cleavage site of acomplement protein; X is NH₂, a blocking group or a detectable label;and Y is COOH or a detectable label, provided that when X is NH₂ or ablocking group, Y is not COOH and when Y is COOH, X is not NH₂ or ablocking group.

Preferably, the complement protein is C4.

Preferably, the C4 protein is human C4 and the cleavage site comprisesArg756.

In a further preferred embodiment, X is a quencher molecule and Y is afluorescent label, or vice-versa, such that a fluorescent signal isobtained when the substrate is cleaved.

In a further aspect, the present invention provides for the use of apeptide of the invention in a method of determining the presence of MASPactivity in a sample.

In yet another aspect, the present invention provides a method ofdetermining the presence of MASP activity in a sample which methodcomprises contacting the sample with a peptide according to theinvention and determining whether said peptide has been cleaved.

In a further aspect, the present invention provides a method ofproducing a pharmaceutical composition of the invention which methodcomprises:

-   (i) providing a complex of non-recombinant MBL and one or more    MASPs;-   (ii) incubating the complex in a suitable buffer to dissociate the    MBL from the one or more MASPs;-   (iii) separating the MBL from the one or more MASPs;-   (iv) screening the MBL obtained from (iii) for MASP activity using a    method of the invention; and-   (v) admixing the resulting purified MBL with a pharmaceutically    acceptable carrier or diluent.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1: Plots of MBL levels against C4 deposition for MASP-depleted MBLand MBL-MASP complex demonstrating superior in vitro MASP2 recruitmentand complement activation by MASP-depleted fractions. C4 results atexcess are plotted at value of 1.0.

FIG. 2: Plots of MBL levels against C4 deposition for MASP-depleted MBLand MBL-MASP complex demonstrating superior in vitro MASP2 recruitmentand complement activation by MASP-depleted fractions. C4 results atexcess are plotted at value of 1.0.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art (e.g. in cell culture, molecular genetics, nucleic acidchemistry, hybridization techniques and biochemistry). Standardtechniques are used for molecular, genetic immunology and biochemicalmethods (see generally, Sambrook et al, Molecular Cloning: A LaboratoryManual, 3rd ed. (2001) Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology(1999) 4^(th) Ed, John Wiley & Sons, Inc.—and the full version entitledCurrent Protocols in Molecular Biology, Ed Harlow and David Lane(editors) Antibodies: A Laboratory Manual, Cold Spring HarbourLaboratory, (1988), and J. E. Coligan et al. (editors) Current Protocolsin Immunology, John Wiley & Sons (including all updates until present),which are incorporated herein by reference) and chemical methods.

A. Purification of MBL Substantially Free of MASPs.

Purified MASP-depleted MBL according to the present invention isobtained from non-recombinant sources—i.e. by purification from animalor human biological material such as plasma. However, the MBL present insuch material is complexed with MASPs and therefore the purification ofMBL substantially free of activated MASPs according to the inventionrequires the separation of MBL from those MASPs.

The purification process typically involves two major steps—thepurification of MBL-MASP complex from other biological material and thedissociation of MBL-MASP complexes to obtain purified substantiallyMASP-depleted MBL. These two steps can occur in any order or even at thesame time. However, typically a pre-purification step is performed toremove at least some biological material, such as non-MBL-MASP plasmaproteins, prior to the dissociation step, and to enrich for MBL-MASPcomplexes since MBL often constitutes less then 0.05% of the totalprotein content of plasma. Thus the biological material, such as bloodplasma, is typically treated to obtain a partially purified compositioncomprising MBL-MASP complex.

One starting point for MBL purification is blood, blood plasma, liverand liver cell cultures. However, MASP-depleted MBL can also be purifiedfrom plasma-derived products or by-products. Preferably, the source ofMASP-depleted MBL is from a side fraction from plasma fractionationprocesses. Examples include, but are not limited to, precipitates orsupernatants from precipitation processes, or filtrates, or sidefractions from ion exchange chromatography, or side fractions fromaffinity chromatography, or fractions from other processes which are notused to produce other plasma based products. As the skilled addresseewould be aware, there are many different known plasma fractionationprocesses. However, the skilled addressee can readily screen for MBL,for example performing mannan binding, MBL antigen or C4 depositionassays and/or affinity chromatography purification of MBL on differentfractions as described herein, to determine which fractions of a givenplasma fractionation process comprises MBL.

An example of a side fraction from plasma fractionation processes as asource of MASP-depleted MBL is crude plasma protein fractions fromindustrial scale ethanol fractionation procedures, such as Cohnfractions II and/or m. These fractions, which include MBL-MASP complexcontaining paste derived from Cohn supernatant I (referred to herein as“euglobulin paste”) are usually discarded and therefore they areeconomically advantageous as a starting material. This is because bloodis a valuable and rare resource and it is therefore desirable tomaximise the use of such side fractions.

The source of MASP-depleted MBL may be from animals or humans. However,it is preferred to purify MASP-depleted MBL from human sources.

Where plasma/plasma by-products are used, they are generally treated toenrich for plasma proteins. Typically, plasma proteins are obtained fromthe plasma or plasma-derived products etc. by a precipitation process.Plasma proteins can be precipitated from plasma or plasma by-productsusing a variety of suitable agents known in the art including variousmolecular weight forms of poly(ethylene glycol), ethanol and ammoniumsulphate.

Further optional steps include filtration, such as depth filtration, anddelipidation.

For example, a euglobulin paste may be obtained as follows: thawedfreshly frozen plasma is treated with water for injection (WFI) and coldethanol at a temperature of below 5° C. The resulting precipitate isthen separated by centrifugation. Typically, the supernatant isdelipidated to adsorb lipoproteins and clarified by filtration. Thesupernatant is then diafiltered using ultrafiltration membrane with anominal molecular weight cut off of not less than 10 000 Daltons tolower the conductivity. The pH of the diafiltered supernatant is loweredto promote euglobulin precipitation and the clarified supernatant isrecovered by filtration. Euglobulin paste is collected during thisprocess.

MBL-MASP complexes are typically extracted from other plasma proteins byaffinity purification, which separates the MBL (most of which iscomplexed to MASP) from other plasma proteins. Generally, the affinitycapture ligand is a sugar. Examples include, but are not limited to,mannan and N-acetylglucosamine. In a preferred embodiment, the affinitycapture ligand mannan e.g. mannan-Sepharose or mannan-agarose. WhereMBL-MASP complexes are present in a precipitate, such as euglobulinpaste, the precipitated proteins are re-solubilised prior to loadingonto the affinity resin. A suitable solubilisation buffer isTris/NaCl/CaCl₂ buffer. For example, euglobulin paste can be solubilisedin a Tris/NaCl/CaCl₂ buffer for 1 hour at room temperature.

Non-solubilised material is generally removed by centrifugation and/orfiltration. Solubilised plasma protein precipitate is loaded onto theaffinity resin and the resin washed prior to elution of MBL-MASPcomplexes with a calcium ion chelating agent, such as EDTA.

An alternative method for purifying MBL-MASP complexes is described inWO99/64453 which uses a polysaccharide matrix, without any conjugatedcarbohydrate ligands such as mannan. Since MBL can bind directly to thepolysaccharide matrix, purification can be effected in a similar mannerto mannan affinity resins but without the need to prepare a conjugatedaffinity resin.

MBL-MASP complex containing solutions, obtained as described above or byother suitable means, are then treated to dissociate the MBL complexi.e. to dissociate MASPs from the MBL. For example, this can be achievedby incubating the MBL complex in a suitable buffer comprising sodiumacetate buffer (pH 4.0-5.0) and EDTA In addition, the buffer may furthercomprise NaCl. Suitable concentrations of NaCl for the dissociation ofMBL-MASP complexes can readily be determined using techniques known inthe art. In one embodiment, the buffer has an NaCl concentration of atleast 0.5 M. More preferably, the buffer has an NaCl concentration ofabout 1 M.

Purified MASP-depleted MBL is then obtained by a suitable purificationstep to separate the MBL from MASPs. Separation is typically on thebasis of size/molecular weight, e.g. size exclusion chromatography,filtration and/or electrophoresis, or on the basis of charge eg. ionexchange chromatography, but other suitable means may be employed. Forexample, Sephacryl S-300 size exclusion chromatography or filtration maybe used. MBL containing fractions are collected and typicallyconcentrated.

Throughout the purification process, it may be desirable at one or morestages to include a concentration step to increase the concentration ofMASP-depleted MBL and/or MBL-MASP complexes. This is achieved by theaffinity purification step but in addition, one or more ultrafiltrationsteps may be included. Preferably, the membranes used forultrafiltration have a molecular weight cutoff of from 10,000 Da to100,000 Da. It is generally desirable to maintain the MBL and/or MASPcomplexes in a compatible buffer during the concentration steps.

It is preferred during the MASP-depleted MBL purification steps toinclude one or more viral inactivation steps since the MASP-depleted MBLis obtained from animal/human biological products. Viral inactivationtechniques are known in the art and typically comprise contacting theMBL with a virus-inactivating agent such as a detergent/solventcombination. Suitable detergents are described in U.S. Pat. No.4,314,997 and U.S. Pat. No. 4,315,991 and include Triton X-100 and Tween80. Suitable solvents include di- and trialkylphosphates such astri(n-butyl) phosphate.

By an “isolated” non-recombinant MBL we mean non-recombinant MBL whichis at least partially separated from molecules with which it isassociated or linked in its native state. Preferably, the isolatednon-recombinant MBL is at least 50% free, preferably at least 75% free,and more preferably at least 90% free from other components with whichit is naturally associated.

The present inventors have shown that the removal of MASPs bound tonon-recombinant MBL enhances the ability of MBL to activate thecomplement pathway. As the skilled addressee would be aware, the removalof any activated MASPs bound to MBL during purification will enhance theactivity of the MBL component. Naturally, the more bound activated MASPsthat are removed the more active the MBL component will be. Accordingly,the present invention extends to any pharmaceutical composition whichhas higher ratios of non-recombinant MBL to activated MASPs whencompared to the staring source (for example plasma).

In one aspect the invention provides a composition comprising purifiednon-recombinant MBL substantially free of activated MASPs, particularlyactivated MASP-2. In this context, the term “substantially free” meansthat a composition comprising 5 μg of isolated non-recombinant MBLprovides a C4 deposition assay result of greater than about 0.3 U/μl.Preferably, the C4 deposition assay result is greater than about 0.5U/μl, more preferably greater than about 0.75 U/μl, more preferablygreater than about 1 U/μl, more preferably greater than about 1.25 U/μl,and even more preferably greater than about 1.5 U/μl. Suitable C4deposition assays are known in the art and described herein.

In another aspect the present invention provides methods of purifyingnon-recombinant MBL substantially free of activated MASP. In thiscontext, and in one embodiment, the term “substantially free” isdetermined as outlined above using a C4 deposition assay. Alternatively,the term “substantially free” can be determined when comparing the MASPactivity of the starting material, namely before any method stepdissociating MBL-MASP complexes, to the MASP activity of the at leastpartially purified MBL product. In a preferred embodiment, the MASPactivity is reduced by at least 75%, more preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 95%, more preferably at least 97%, and even more preferably atleast 99%. MASP activity assays are known in the art and include thosedescribed herein.

The term “activated MASP” means that the MASP is not in its pro-enzymeform but is in its active form, due to proteolysis. Activated MASPs canbe distinguished from pro-enzyme by size—the pro-enzyme has a molecularweight of about 70 to 110 kDa whereas the activated enzyme consists of aheavy chain of about 50 to 65 kDa and a light chain of about 30 to 40%Da. These can be resolved by gel electrophoresis under reducingconditions followed by visualisation and/or immunodetection (e.g.Western blotting).

However, in a preferred embodiment, the MBL is substantially free ofMASPs, whether activated or not as calculated relative to MBL-MASPcomplex.

MBL functions more effectively as an oligomer due to increased avidity.Consequently, it is preferred that the purified MASP-depleted MBL of theinvention remains in its native oligomeric state. Preferably at least50% of the purified MBL is present as oligomers, more preferably astetramers or higher order oligomers.

B. Pharmaceutical Compositions

Purified MASP-depleted MBL according to the invention may be combinedwith various components to produce MASP-depleted MBL compositions. Thecompositions typically comprise a pharmaceutically acceptable carrier ordiluent to produce a pharmaceutical composition (which may be for humanor animal use) to produce a pharmaceutical composition of the invention.The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that do not produce an allergic, toxic, or otherwiseadverse reaction when administered to an animal, particularly a mammal,and more particularly a human. Pharmaceutically acceptable media orcarriers include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, stabilizers, isotonic andabsorption delaying agents and the like. The use of such media andagents for pharmaceutical active substances is well known in the art.

Suitable carriers and diluents include isotonic saline solutions, forexample phosphate-buffered saline. The carrier can also be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), suitable mixtures thereof and vegetable oils. The properfluidity can be maintained, for example, by the use of a coating, suchas lecithin, by the maintenance of the required particle size in thecase of dispersion and by the use of surfactants. The prevention of theaction of microorganisms can be brought about by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminium monostearate and gelatin.

Examples of suitable stabilizers include, but are not limited to,pharmaceutical grades of a monosaccharide, a disaccharide, sucrose,lactose, trehalose, mannitol, sorbitol, inositol, dextran and the like;plasma protein products other than MBL or MASP such as albumin; aminoacids; and polyols (for example, polyethylglycol) and the like.

The composition may be in any suitable form such as a liquid or a solid.Solid compositions may be obtained using any technique known in the artincluding spray-drying, freeze-drying, spray-freeze drying, air-drying,vacuum-assisted drying, fluid bed drying and the like.

The composition of the invention may be administered by directinjection. The composition may be formulated for various routes ofadministration including parenteral, intramuscular and intravenousadministration. Other delivery systems can include time-release, delayedrelease or sustained release delivery systems. Such systems can avoidrepeated administrations of the MASP-depleted MBL compositions of theinvention, increasing convenience to the subject and the physician. Manytypes of delayed release delivery systems are available and known tothose of ordinary skill in the art. They include polymer-based systemssuch as polylactic and polyglycolic acid, polyanhydrides andpolycaprolactone; nonpolymer systems include lipids such as sterols, andparticularly cholesterol, cholesterol esters and fatty acids or neutralfats such as mono-, di and triglycerides; hydrogel release systems;silastic systems; peptide based systems; wax coatings, compressedtablets using conventional binders and excipients, partially fusedimplants and the like. In addition, pump-based hardware delivery systemscan be used, some of which are adapted for implantation.

A long-term sustained release implant also may be used. Long-termrelease, as used herein, means that the implant is constructed andarranged to deliver therapeutic levels of purified MBL for at least 30days, and preferably 60 days. Long-term sustained release implants arewell known to those of ordinary skill in the art.

Typically, MBL protein may be administered at a dose of from 0.001 to100 mg/kg body weight, preferably from 0.01 to 10 mg/kg, more preferablyfrom 0.05 to 1 mg/kg body weight.

For example, a suitable initial dose for an MBL deficient adult is 6 mgMBL in 100 ml saline, given as an infusion, with follow up doses ofabout 6 mg twice weekly as required.

The routes of administration and dosages described are intended only asa guide since a skilled practitioner will be able to determine readilythe optimum route of administration and dosage for any particularpatient and condition.

Compositions of the present invention may be co-administered withcompositions comprising unactivated purified MASPs. Such MASPs suitablefor co-administration may be obtained recombinantly using techniquesknown in the art. The nucleotide sequence of human MASP-1 is availableas GenBank Accession No. NM_(—)001879. The nucleotide sequence of humanMASP-2 is available as GenBank Accession AH010229. The nucleotidesequence of human MASP-3 is available as GenBank Accession AF284421.

C. Assays for MASP Activity

It is desirable to assay the MBL compositions of the invention toconfirm that they are substantially free of MASP. The presence of MASPscan be measured using a variety of techniques including immunologicalmethods (e.g. BLISA). In addition, MASP activity can be determined usingassays based on cleavage of labelled substrates—such as labelledpeptides derived from the C-terminus of the products of MASP cleavage ofcomplement proteins—C2, C3, C4 or C5.

Further, we have developed a highly sensitive assay method for activeMASP based on the use of substrates derived from the MASP cleavage siteon the C4 protein. These substrates differ from those disclosed in U.S.Pat. No. 6,235,494 because they contain amino acid sequences from bothsides of the cleavage site of the uncleaved complement protein whereasthe substrates of U.S. Pat. No. 6,235,494 do not contain any amino acidsresidue C-terminal of the arginine cleavage site. The inclusion of theadditional amino acids, such that the arginine is flanked by aminoacids, provides additional specificity and reliability.

Accordingly, the present invention provides a peptide of formulaX-R1-Arg-R2-Y wherein R1-Arg-R2 is a peptide consisting of 6 or morecontiguous amino acids derived from the MASP cleavage site of acomplement protein; X is NH₂, a blocking group or a detectable label;and Y is COOH or a detectable label provided that when X is NH₂, or ablocking group, Y is not COOH and when Y is COOH, X is not NH₂ or ablocking group.

Preferably R1 and/or R2 comprise at least three amino acids, preferablyat least four amino acids. Preferably R1-Arg-R2 comprises fewer than 10amino acids. More preferably R1-Arg-R2 consists of 7 or 8 amino acids.

The complement protein cleavage site from which R1-Arg-R2 is derived ispreferably the MASP cleavage site of a C2, C3, C4 or C5 protein, such asArg756 of human C4 (Accession No. P01028).

Examples of peptides of the invention include, but are not limited to:X-Lys-Gly-Gly-Leu-Gln-Arg-Ala-Leu- (SEQ ID NO:1) Glu-Ile-YX-Gly-Leu-Gln-Arg-Ala-Leu-Glu-Ile-Y (SEQ ID NO:2)X-Gly-Gly-Leu-Gln-Arg-Ala-Leu-Glu-Y (SEQ ID NO:3)X-Gly-Gly-Leu-Gln-Arg-Ala-Leu-Glu- (SEQ ID NO:4) Ile-YX-Glu-Ser-Leu-Gly-Arg-Lys-Ile-Gln- (SEQ ID NO:5) Ile-Gln-YX-Ser-Leu-Gly-Arg-Lys-Ile-Gln-Ile-Y (SEQ ID NO:6)X-Glu-Ser-Leu-Gly-Arg-Lys-Ile-Gln-Y (SEQ ID NO:7)X-Ser-Leu-Gly-Arg-Lys-Ile-Gln-Ile- (SEQ ID NO:8) Gln-Y

Derivatives of naturally occurring complement protein cleavage sitesequences may also be used. The term “derivatives” means that minorsubstitutions, insertions and deletions may be made to the naturallyoccurring complement protein cleavage site sequences, other than thearginine residue, provided that the resulting sequences can be cleavedby one or more MASPs and that at least two, preferably at least three orat least four, amino acid residues are present C-terminal of thearginine cleavage site.

Conservative substitutions may be made, for example according to theTable below. Amino acids in the same block in the second column andpreferably in the same line in the third column may be substituted foreach other: ALIPHATIC Non-polar G A P I L V Polar-uncharged C S T M N QPolar-changed D E K R AROMATIC H F W Y

Substitutions may also include the use of non-naturally occurring aminoacid analogues.

Considering the disclosure herein, the skilled addressee could readilyscreen derivates (either comprising naturally and/or non-naturallyoccurring amino acids) of labelled known MASP cleavage substrates todetermine suitable derivates which could be used in the assays of thepresent invention.

At least one of X or Y are detectable labels that permit detection ofcleavage of the substrate. Any suitable detectable labels may be used,such a radiolabels, colorimetric, bioluminescent, chromogenic orfluorescent labels. However, in a preferred embodiment, one of thelabels is a fluorescent label. In a highly preferred embodiment, X is afluorescent label and Y is a quencher molecule, or vice-versa. In thisway, provided that X and Y are within a certain distance of each other(e.g. 8 or less amino acids apart) in the uncleaved substrate, nofluorescence signal will be obtained. However on cleavage of thesubstrate by a MASP, the quencher molecule will be separated from thefluorescent label and fluorescent signal will be obtained.

Examples of quencher molecules include, but are not limited to,dinitrophenyl ethylenediamine (EDDnp) and Lys(Dnp). Examples offluorescent labels include, but are not limited to,7-amino-4-methylcoumarin (AMC) and aminobenzoic acid (Abz). Examples ofcolourimetric molecules include, but are not limited to,para-nitroaniline.

Peptides of the invention are typically made by synthetic means usingtechniques well known to skilled persons such as solid phase synthesis.Various techniques for chemically synthesising peptides are reviewed byBorgia and Fields (2000) and described in detail in the referencescontained therein.

The assay systems herein may be provided in kit form that is useful fordetermining activated MASP levels in a sample. The kits may include asubstrate contained in a suitable container or linked to a solidsupport, such as a microtiter plate or other suitable support, orcontained in the wells of a microtiter plate. Kits may also includeinstructions for performing the assays.

The kits will optionally include other reagents for performing theassays, including controls, trypsin, Futhan or other serine proteaseinhibitor, buffers, such as PBS, stop solutions, and other suchreagents. The kits may also include suitable ancillary supplies, such asmicrotiteir plates, vials, labeled ligand or labeled anti-ligand,calibrator solutions, controls, wash solutions, solid-phase supports andthe like.

The peptides of the invention can be used to assay for MASP activity ina sample, such as a sample containing MASP-depleted MBL purified asdescribed above. Samples may also include biological samples, such asblood samples, from patients, including patients suspected of having anMBL deficiency.

Accordingly, the present invention provides a method of determining thepresence of MASP activity in a sample which method comprises contactingthe sample with a peptide of the invention and determining whether saidpeptide has been cleaved.

The method for detection of proteolytic activity, i.e. cleavage of thesubstrate, will vary depending on the type of label. Detection can, forexample, be based on quantitative or qualitative measurements.

For quantitative measurements, typically the signal emitted by the labelis measured from the beginning of the reaction and the results used toobtain an initial rate. Substrates consisting only of the residuesN-terminal to the cleavage site of C4 show normal Michaelis-Mentenkinetics for their cleavage by both C1s and the MBL-MASP complex. Thismeans that the dependence of the initial velocity for the cleavagereaction on substrate concentration can be described by a rectangularhyperbola and the constants K_(m) (Michaelis constant which equates tothe affinity between enzyme and substrate) and V_(max) (maximal velocityof the reaction) can be derived from a non-linear regression fit of thedata.

Substrates which incorporate amino acid residues both N- and C-terminalto the cleavage point of C4, do not show Michaelis-Menten kinetics forthe cleavage of the substrate by C1s and MBL-MASPs, however. Instead,the dependence of initial velocity for the cleavage on substrateconcentration is best described by a sigmoidal curve. This indicatesthat the enzyme is displaying allosteric behaviour or positiveco-operativity in the cleavage of P₄-P₄′ substrates. Non linearregression fitting of the curve in this case yields three differentconstants: V_(max) (again, the maximal velocity of the interaction),K_(0.5) (or the substrate concentration at half V_(max), which indicatesthe affinity between enzyme and substrate) and the Hill constant (h,indicating the degree of positive co-operativity).

It has been reported previously that C1 inhibitor (C1INH) binds to theMASPs at a ratio of 1:1. Thus a preparation of C1INH of known activeconcentration can be used to titrate the amount of active enzyme in MASPpreparations. This can be carried out using C1s as a positive control.This will then allow the calculation of k_(cat) for the interactionbetween MASPs and fluorometric substrates. Once activity (fluorescence)is plotted against C1INH concentration, the active enzyme concentrationof the MASP preparation can be determined as the point at which the lineintercepts the x-axis.

The K_(0.5) and V_(max) values for an enzyme substrate reaction can thenbe determined using allosteric kinetics. Knowledge of the active enzymeconcentration then allows calculation of the k_(cat) constant for theenzyme substrate reaction using the following equation:k _(cat) =V _(max)/[active enzyme]

Where V_(max) is the maximal velocity of the enzyme-substrate reactionand [active enzyme] is the molar amount of enzyme present in thepreparation that is capable of cleaving substrate.

Once the k_(cat) value has been determined, MASP preparations can beassayed at substrate concentrations twice the K_(0.5) value, yielding avelocity that is nominally equivalent to V_(max). The k_(cat) andV_(max) values can then be substituted into the equation:k_(cat)=V_(max)/[active enzyme]. Rearrangement of the equation willyield an estimate of the active enzyme concentration in the sample.

D. Therapeutic Uses

The pharmaceutical compositions of the present invention may be used totreat subjects in need of MBL.

As used herein, an “effective amount” means an amount sufficient to atleast increase the ability of the subjects immune system to opsonisepathogens and induce the complement cascade in response to the pathogen.

Subjects include bone marrow allograft recipients, subjects with cysticfibrosis, subjects with an immunodeficiency, subjects with acutelymphoblastic leukaemia, subjects with community acquired or nosocomialsepticaemia, subjects with or susceptible to an infection by a pathogen,low birthweight and/or premature infants. Typically, the subject has anMBL deficiency, such as congenial MBL deficiency.

As used herein, an MBL deficiency is where the subjects MBL levels arebelow 500 ng/ml and/or the subjects C4 deposition assay result is lessthan 0.3U/ul. In particular individuals having an MBL level below 400ng/ml will benefit from the methods of the invention, such asindividuals having an MBL level below 300 ng/ml, or such as individualshaving an MBL level below 250 ng/ml, or such as individuals having anMBL level below 200 ng/ml.

The pathogen may be any organism which comprises a molecule to which MBLbinds resulting in activation of a complement pathway. Such pathogensmay be yeast, gram negative enteric bacteria, gram positive bacteria,mycobacteria, some viruses, and certain parasites. More specificexamples of such pathogens include, but are not limited to, thoseselected from the group consisting of: Parasites such as Cryptospridiumparvum and Plasmodium falciparum; Fungi such as Cryptococcus sp.including Cryptococcus neoformans, Candida albican and Aspergillusfumigatus; and Bacteria such as beta haeniolytic streptococcus group A,Bifidobacterium bifidum, Actinomyces israelli, Proprionibacterium acnes,Bacteroides sp., Escherichia coli, Eubacterium sp., Fusobacterium sp.,Veillonella sp., Haemophilus influenzae, Neisseria gonorrhoeae,Neisseria meningitidis, Staphylococcus aureus, Salmonella enterica,Burkholderia cepacia and Klebsiella pneumoniae.

Particular indications include: Neurology: Chronic inflammatorydemyelinating polyneuropathy (CIDP), Multifocal motoric neuropathy,Multiple sclerosis, Myasthenia Gravis, Eaton-Lambert's syndrome, OpticusNeuritis, Epilepsy; Gynaecology: Abortus habitualis, Primaryantiphospholipid syndrome; Rheumatology: Rheumatoid arthritis, Systemiclupus erythematosus, Systemic scleroderma, Vasculitis, Wegner'sgranulomatosis, Sjogren's syndrome, Juvenile rheumatoid arthritis;Haematology: Autoimmune neutropenia, Autoimmune haemolytic anaemia,Neutropenia; Gastrointestinal: Crohn's disease, Colitis ulcerous,Coeliac disease; Others: Asthma, Septic shock syndrome, Chronic fatiguesyndrome, Psoriasis, Toxic shock syndrome, Diabetes, Sinuitis, Dilatedcardiomyopathy, Endocarditis, Atherosclerosis, Adults with AIDS andbacterial infections, Primary hypo/agammaglobulinaemia including commonvariable immunodeficiency, Wiskot-Aldrich syndrome and severe combinedimmunodeficiency (SCID), Secondary hypo/agammaglobulinaemia in patientswith chronic lymphatic leukaemia (CLL) and multiple myeloma, Childrenwith AIDS and bacterial infections, Acute and chronic idiopathicthrombocytopenic purpura (ITP), Allogenic bone marrow transplantation(BMT), Kawasaki's disease, and Guillan-Barre's syndrome.

It has been shown that a deficiency in MBL predisposes infants to acutelymphoblastic leukaemia. Consequently, the methods of the invention mayalso be used prophylactically to prevent disorders caused by/associatedwith MBL deficiency, such as acute lymphoblastic leukaemia. Thus,subjects also include those at risk of developing any of the abovedisorders, as appropriate, due to an MBL deficiency, such asMBL-deficient infants at increased risk of developing acutelymphoblastic leukaemia.

The present invention will now be described further with reference tothe following Examples, which are illustrative only and non-limiting.

EXAMPLES Example 1 Purification of MASP-Depleted MBL

Fresh frozen plasma was softened and thawed at temperatures below 5° C.and the cryoprecipitate separated from the cryosupernatant by continuousflow centrifugation. Cold ethanol was added to the cryosupernatant to afinal concentration of 8% (v/v). The precipitate formed was separatedfrom the supernatant by centrifugation or filtration at −2° C.±1° C. Thesupernatant was treated to adsorb lipoproteins and clarified byfiltration. Delipidated supernatant was diafiltered usingultrafiltration membrane with nominal molecular weight cut off of notless than 10 000 Daltons to lower the conductivity. The pH of thedelipidated diafiltered supernatant was lowered to promote euglobulinprecipitation and the clarified supernatant recovered by filtration. Theeuglobulin paste collected during this process was further purified toextract MBL-MASP complex.

The purification process was carried out at ambient temperature.Euglobulin paste was solubilised in a 20 mM Tris/100 mM NaCl/15 mM CaCl₂buffer for 1 hour at room temperature. Non-solubilised material wasremoved by centrifugation. Affinity chromatography was employed toseparate the MBL-MASP complex from other plasma proteins. Solubilisedeuglobulin paste was loaded onto a mannan-agarose column. The column waswashed with a Tris/NaCl/CaCl₂/Tween 20 buffer before the MBL-MASPcomplex was eluted with 10 mM EDTA.

The eluate was then incubated in 0.1 M sodium acetate buffer (pH 5.0)containing EDTA to dissociate MASPs from the MBL molecules. The materialwas then applied to a sephacryl S-300 size exclusion column andfractions analysed by SDS page. Results showed separation of MBL fromother protein components. Fractions were analysed for MASP activity inthe substrate assays as described in this document. Seven fractionscontained MBL with low levels, or near-depleted, of MASP activity. Totalprotein concentration of each fraction was in the range of 10-90 μg/mL.

Example 2 Assays to Confirm Absence of MASPs

Pooled MBL fractions from Example 1 are tested to confirm that the MBLis substantially free of MASPs.

Substrate Design

Substrates were designed for MASPs based on the amino acids surroundingthe cleavage site (⁷⁵⁶R) of the natural substrate, C4 protein. Thesesubstrates are used to determine the activity of the MASPs in the MBLpurified material.

The present inventors have found that the inclusion of additional aminoacid such that the arginine is flanked by amino acids, providesadditional specificity and reliability (Table 1). TABLE 1 Kineticconstants for the proteolytic activity of MASPs in purified MBL- MASPcomplex on synthetic substrates based on the P₄-P₁ and P₄-P₄′ amino acidof complement protein C4 Affinity constant Substrate K_(m) (μM) K_(0.5)(μM) C4 (P₄-P₁) 198.0 ± 20.4 — C4 (P₄-P₄′) — 6.50 ± 0.32

The substrate (2Abz-GLQRALEI-Lys(Dnp)-NH₂) includes the four amino acidsbefore and after the C4 cleavage site and an aminobenzoic acid (Abz)fluorescent group attached to its N-terminal end. The Abz group isquenched by Lys(Dnp), when located no more than 8 amino acids away fromthe Abz group. The cleavage site of the substrate is located between theAbz group and the Lys(Dnp) group, so that when the enzyme cleaves thesubstrate, the quenching ability of Lys(Dnp) is lost and the Abz groupis able to fluoresce. The change in fluorescence can then be measuredand is proportional to the proteolytic activity of the enzyme. It hasbeen demonstrated that MASPs present in the purified MBL material cleavethis substrate (K_(0.5)=6.5 I.

If proteolytic activity (i.e. a change in fluorescence) is observed whenassaying the purified MBL material, this indicates that the MASPs havenot been successfully removed. ELISA or immunoblot using anti-MASPantibodies is then conducted to confirm that this finding is attributedto the proteolytic activity of the MASPs and not some other protease inthe MBL product. C1s, the closest homologue of the MASPs can be used asa positive control for the substrate cleavage assays.

Substrate Cleavage Assays

The substrate is diluted in fluorescent assay buffer (FAB—50 mMtris-hydroxymethylene, 150 mM NaCl, 0.2% polyethylene glycol 8000, 0.02%sodium azide, pH 7.4) so that a final concentration equal to V_(max) isachieved. The substrate and enzymes (C1s (10 μg/mL) or purified MBLmaterial) are incubated for several minutes in a fluorescence platereader set at 37° C. 100 μL of diluted substrate is then transferredinto wells containing 100 μL diluted enzyme (C1s or purified MBLmaterial) and the kinetics of fluorescence is measured as follows:excitation=320 nm; emission=420 nm. Each test is performed intriplicate. The amount of fluorescence is then read off the standardcurve to calculate the concentration of active MASP enzymes in thepurified MBL material.

Example 3 MASP-Depleted MBL is Capable of Recruiting MASP from Plasmaand Activating the Complement Cascade

Standard Curve and Control Material for Quantitation Assays

All quantitation assays were standardised using an international,primary standard pool serum (Statens Serum Institut, Copenhagen,Denmark), containing 3.3 μg MBL/ml serum. For the sandwich ELISA and theC4 deposition assay, a standard curve was made with 1:25, 1:50, 1:75,1:100, 1:150 and 1:200 dilutions of this serum, tested in triplicate.Standard dilutions for the mannan binding ELISA were 1:25, 1:50, 1:100,1:150, 1:200, 1:300 and 1:400. Diluents were as detailed below. Anin-house secondary control was prepared from pooled normal donor plasmaand run in triplicate on each test plate, the results plotted for eachrun. Results of any test runs, in which values obtained for the in-housecontrol MBL were outside +/−2SD from the previously determined meanvalue, led to rejection of the whole run. Run to run standard curveswere overlayed to ensure a constant slope and thus provide anothersensitive means of quality control.

Quantitation of MBL by Double Antibody Sandwich ELISA (“Double AntibodyAssay”)

This MBL antigen detection assay was based on the original method ofGarred et al. (1992) except that a commercial IgG mouse monoclonal,anti-human MBL, which targets a peptide epitope in the coliagenous neckregion of the MBL structural unit, was used instead of rabbit polyclonalanti-MBL.

Briefly, flat-bottomed microtitre plates (Nunc-Immuno Maxisorp, NalgeNunc International) were coated overnight at 4° C. with 2 μg/mlmonoclonal anti-MBL (Staten Serum Institut, Copenhagen, Denmark) infresh 50 mM carbonate-bicarbonate buffer, pH 9.6. Normal donor plasmaswere tested in triplicate, diluted to 1:25 and 1:100 in 0.1 M PBS-0.05%Tween 20, pH 7.4 (IBST), which was also the wash buffer. After 90minutes at 22° C., wells were washed 3 times and monoclonal anti-MBLbiotinylated using Biotin Tag (Sigma-Aldrich Pty Ltd) was added at1:4000, this dilution determined by chequerboard titration with poolednormal plasma. After 90 minutes at 22° C., wells were washed 3 times andExtrAvidin peroxidase conjugate at 1:500 was added for 40 minutes at 22°C. Colour was developed with OPD tablets and diluent (AbbottLaboratories, Illinois, USA), stopped with 1N H₂SO₄ and read immediatelyat 490 nm in a Bio-Rad plate reader (Bio-Rad laboratories Pty Ltd.,Regents Park, Australia).

Between run coefficients of variation (CV) were 8.2% at 1:25 and 12% at1:100. Run to run standard curves were overlayed to ensure a constantslope and thus provide another sensitive means of quality control.

Quantitation of MBL by Mannan Binding ELISA (“Mannan Biniding Assay”)

This assay measures the ability of MBL to bind to mannan coated onto apolypropylene matrix, and is based on the method of Holmskov et al.(1993).

Microtitre plates (as above) were coated overnight at 4° C. with 10μg/ml mannan (Sigma-Aldrich Pty Ltd, Castle Hill, Australia) in fresh 50mM carbonate-bicarbonate buffer, pH 9.6. Normal donor plasmas weretested as in the sandwich ELISA but with Tris Buffered Saline with 0.05%Tween-20 (TBST) supplemented with 15 mM CaCl₂, pH 7.5 as diluent andwash buffer. All incubations were at 22° C., the MBL and antibodycapture time were each 90 minutes. Incubation with ExtrAvidin peroxidaserequired only 30 minutes. Colour development and reading was as for thesandwich ELISA.

Between run coefficient of variation (CV) were 6.1% at 1:25 and 8.8% at1:100. Run to run standard curves were overlayed to ensure a constantslope and thus provide another sensitive means of quality control.

The specificity of these assays for MBL was confirmed by the linearstandard curves obtained with the Statens Serum Institut primarystandard pool serum. We also performed limiting dilution testing in eachassay with a purified mannose binding lectin prepared by mannan affinitychromatography and confirmed in the Western immunoblot. In the mannanbinding assay we also were able to block binding of plasma MBL bydiluting test samples in 10 mM EDTA or 0.1 M mannose solution.

Functional Complement (C4) Deposition Assay (“C4 Deposition Assay”)

Originally described for detection of deposited C3b and C3bi by Super etal. (1989) and modified by Valdimarsson et al. (1998), this assaydemonstrates deposition of C4b following activation of MBL by bindingwith solid-phase purified mannan.

Microtitre plates (as above) were coated overnight at 4° C. with 1 μg/mLmannan (Sigma-Aldrich Pty Ltd, Castle Hill, Australia) in fresh 50 mMcarbonate-bicarbonate buffer, pH 9.6. Normal donor plasmas were testedin triplicate, diluted to 1:25 in TBST with 15 mM CaCl₂, pH 7.2 whichwas also the wash buffer. A 1:10 dilution was also tested andinterpreted only to confirm low or near absent levels of C4 depositionin donors with low amounts of MBL. After 90 minutes at 22° C., wellswere washed 5 times and MBL-deficient human serum (completely deficientin MBL, obtained with informed consent) diluted 1:20 in barbital buffer,14 mM NaCl, 10 mM sodium barbitone and 5 mM CaCl₂ was added to wells andincubated at 22° C. for 30 minutes to enable complement activation.Wells were washed 5 times and biotinylated rabbit anti-human C4(Sigma-Aldrich Pty Ltd) biotinylated using Biotin Tag (Sigma-Aldrich PtyLtd) which was added at 1:1500 in TBST. Following incubation at 22° C.for 90 minutes, wells were washed 5 times and 1:500 ExtrAvidinperoxidase (Sigma-Aldrich Pty Ltd) in TBST was added and incubated at22° C. for 40 minutes. Colour development and reading was as for thequantitation assays.

1 μl of Statens Serum Institut (SSI) MBL Standard was arbitrarilyassigned 1 unit of C4 deposition activity. The assay was standardisedagainst the SSI standard. Between run CV for the assay at 1:25 was 9.4%.

Demonstration MASP-Depleted MBL can Recruit MASP and Activate theComplement Cascade

Two fractions of the purified MBL obtained in Example 1 (fractions 3 and5 [40 μg/mL and 80 μg/mL respectively] with both fractions demonstratingMASP activity <10% of the concentration of the MBL-MASP complex—asdetermined using the C4 P₄-P₁ substrate), were chosen for titration andcomparison with affinity purified MBL-MASP complex. Fractionsdemonstrating a slope/sec of less than or equal to 10% of the S300 loadmaterial (concentrated MBL-MASP complex eluate buffer exchanged inacetate buffer) were considered MASP-depleted. The positive control forMASP-2 activity was MASP containing, affinity-purified MBL complex. Allfractions obtained in Example 1 were considered MASP-depleted with MASPactivity measured as slope/sec less than 2.3 (range 0.5-2.3). Theslope/sec for the positive control (MASP containing affinity-purifiedMBL complex) was 22. Both MASP-depleted fractions and the MBL-MASPcomplex were titrated to provide MBL protein concentrations at 1 to 100μg/ml. Fractions were assayed in parallel in the mannan-binding andC4-deposition assays as described in this document. The result from theMannan-binding assay was plotted against C4 activity for each fraction.

Results

Plots of MBL levels against C4 deposition for MASP-depleted MBL andMBL-MASP complex clearly demonstrated superior in vitro MASP2recruitment and complement activation by MASP-depleted fractions (SeeFIG. 1 and Table 2). Results for MBL-MASP complex began to plateau atless than 10 μg/ml MBL. A previous C4 deposition assay on the sameMBL-MASP complex batch gave a C4 result of 0.27 U/μl for 25 μg/mL MBL.This is concordant with the corresponding MBL concentrations in thistitration. This experiment indicates that MASPs activated by affinitypurification remain docked to MBL, blocking approach of fresh MASPs whenMBL binds to mannan. TABLE 2 Titration of MASP-depleted fractions andaffinity purified MBL- MASP complex in the mannan-binding andC4-deposition assays. MASP-depleted MBL-MASP Complex MASP-depleted Frac.5 Frac. 3 MBL ug/mL C4 U/ul MBL ug/mL C4 U/ul MBL ug/mL C4 U/ul 50 0.28980 xs 40 0.321 40 xs 40 xs 20 0.25 30 0.906 30 xs 15 0.246 20 0.762 20xs 11.41 0.204 16 0.805 16 xs 6.94 0.197 12 0.716 12 xs 5.95 0.172 100.671 10.53 0.818 4.42 0.168 8.79 0.465 7.97 0.761 4.08 0.138 7.83 0.4175.65 0.568 3.3 0.197 5.11 0.304 2.57 0.359 2.081 0.103 2.49 0.137 2.10.202 1.032 0.068 1.32 0.081 1.02 0.107 0.624 0.05Results also Plotted in FIG. 1.

Example 4 Confirmation of MASP Recruitment and Complement Activation byMASP-Depleted MBL

The fractions supplied in Example 3 were pooled according to totalprotein concentration. The first pool contained fractions 3-7 and had atotal protein of 85 μg/ml. The second pool was made from fraction 8 and9, this had a total protein concentration of 35 μg/ml. The twoMASP-depleted MBL pools were again titrated in the range of 1-100 μg/mlin parallel with the MBL-MASP complex. All samples were assayed on boththe Mannan-binding and C4-deposition assays in parallel. Actual MBLquantification (mannan-binding assay) results were graphed againstcorresponding C4 activation capability for the fraction.

Results

Pools of MASP-depleted MBL fractions gave reproducible results ascompared to the individuals fractions analysed in Example 3. Complementactivation capacity reached excess in the pooled fraction 3-7 andreached the high assay limit for fractions 8-9, whereas the MBL-MASPcomplex plateaued at 12 μg, and failed to substantially increase C4deposition with increased MBL concentration. This reproduced the findingin Example 3. MASP-depleted MBL had superior ability to recruit MASP andas a result was more efficient at activating the complement cascade thanthe MBL-MASP complex (see FIG. 2 and Table 3). TABLE 3 Titration ofpooled MASP-depleted fractions (3-7 and 8-9) and affinity purifiedMBL-MASP complex in the mannan binding and C4 deposition assays MBL-MASPComplex MASP-depleted Pool 3-7 MASP Pool 8-9 MBL ug/mL C4 U/ul MBL ug/mLC4 U/ul MBL ug/mL C4 U/ul 50 0.308 85 xs 40 0.292 80 xs 35 0.969 200.244 40 0.954 30 0.776 10.998 0.22 20 0.825 20 0.595 7.967 0.203 14.0450.718 12.718 0.552 6.814 0.19 9.377 0.596 10.205 0.48 4.681 0.171 7.7960.55 8.995 0.435 5.382 0.159 7.151 0.506 7.362 0.403 4.011 0.138 5.6170.38 6.506 0.31 2.802 0.114 3.905 0.25 4.278 0.189 2.237 0.104 1.6770.104 2.032 0.108 1.065 0.057 1.124 0.043 1.122 0.049 0.471 0.054Results also Plotted in FIG. 2

CONCLUSIONS

The results of Examples 3 and 4 show that MASP-depleted MBL is able torecruit MASPs from plasma and successfully activate the complementcascade. Free MASPs circulate in the plasma, at levels above that of theMBL-MASP complex. Individual MASP-1 levels range from 1.48 to 12.83μg/mL. The arithmetic mean±s.d. of MASP-1 levels in serum is 6.27+1.85μg/mL. The serum level of MASP-1 has been found to be strongly dependenton age as is the serum MBL level. The serum level of MASP-1 has alsobeen found to be much higher than that of MBL (1.71±1.13 μg/mL), and themajor portion of human serum MASP-1 appears to exist in the circulationas a form unbound to MBL (Terai et al., 1995). MASP-2 levels arebelieved to be lower than MASP-1 levels. When MBL-MASP complex isdisrupted by dialysis against sodium acetate buffer (pH 5.0), and thensubsequently dialysed back into TBS-TEDTA buffer (pH 7.8), MBL and MASPshave been shown to be in complex. The low pH dissociation of MBL-MASPcomplex is reversible (Tan et al., 1996).

Furthermore these data demonstrate that MBL purified as a complex haslimited ability to activate the complement cascade probably due todecreased ability to bind fresh MASPs as MASP binding sites may beblocked by MASP activated during the purification process. For instance,when MBL is purified by affinity purification on mannan columns asdescribed previously or using a non-conjugated polysaccharide matrix astaught in WO99/64453, it co-purifies with MASPs. However, the majorityof MASPs co-eluted with MBL are in their activated form, with only afraction of MASPs remaining in their pro-enzyme (90 kDa) form due tocontact with polysaccharide substrates during the affinity purificationsteps.

Testing of MASP-depleted MBL in the C4 deposition assay demonstrates itssuperior ability to recruit MASP and initiate complement activation invitro compared with purified MBL-MASP complex. Normally, MASPs producedin the body associated with the MBL-MASP complex are only activatedafter specific binding of the MBL to a foreign organism. This serves asthe major point of regulation for the activation of complement by theMBL pathway. Thus administering MBL containing activated MASPseliminates this regulation mechanism.

Physiological inhibitors include C1 inhibitor (C1 INH), which formscomplexes with activated MASP-1 and MASP-2. Also, C3 cleavage by MASP-1is inhibited by C1 INH in a dose dependent manner. This is the same forC2 activation by MASP-1 and C4 & C2 activation by MASP-2. The MASPs arealso inhibited by a2-macroglobulin, which has broad protease inhibitoryactivity (Storgaard et al 1995).

While deficiencies of an inhibitor such as C1 INH may be reasonablyrare, it would also mean that individuals deficient in an inhibitorwould be prone to complications following MBL-MASP complex beingadministered, due to inappropriate activation of the complement cascade.Thus, we consider that MBL purified with associated MASPs attached is aproduct with lowered efficacy and could even have potential clinicaldangers.

All publications mentioned in the above specification are hereinincorporated by reference.

Various modifications and variations of the described methods and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are apparent to those skilled in molecular biology orrelated fields are intended to be within the scope of the invention.

Throughout this specification, unless the context requires otherwise,the word “comprise”, and variations such as “comprises” and“comprising”, will be understood to imply the inclusion of a statedinteger or step or group of integers or steps but not the exclusion ofany other integer or step or group of integers or steps.

Where specific embodiments are described in particular sections above,the embodiments apply mutatis mutandis to other sections as appropriate.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

REFERENCES

-   Anderson, O. et al. (1992) Scandinavian J. Immunol. 36:131-41.-   Borgia, J. A and Fields, G. B. (2000) Trends Biotech. 18:243-51.-   Garred, P. et al. (1992) Eur. J. Immunogenet. 19:403-12.-   Holmskov, L. et al. (1993) Glycobiology 3:147-53.-   Kilpatrick, D. C. (2000) Human Reprod. 15: 94143.-   Koch, A, et al. (2001) JAMA 285:1316-21.-   Koppel, R et al. (1994) J. Chromatography 662:191-6.-   Matsushita, M. et al. (1992) J. Exp. Med. 176:1497-502.-   Storgaard, P. et al. (1995) Scan. J. Immunol. 42:373-80.-   Super, M. et al. (1989) The Lancet 2:1236-9.-   Tan, S. M. et al. (1996) J. Biochem. 319:329-32.-   Terai, I. et al. (1995) Int. Immunol. 110:317-23.-   Turner, M. W. (1996) Immunol. Today 17:532-40.-   Valdimarsson, H. et al. (1998) Scandinavian J. Immunol. 48:116-23.

1. A pharmaceutical composition comprising isolated non-recombinantmannose binding lectin (MBL) substantially free from activated MBLassociated serine proteases (MASPs) together with a pharmaceuticallyacceptable carrier or diluent.
 2. A composition according to claim 1,wherein the MBL is substantially free from MASPs.
 3. A compositionaccording to claim 1 or claims 2, wherein the MBL is human MBL.
 4. Acomposition according to any one of claims 1 to 3, wherein the MBL isobtained by a method comprising: (i) providing a complex ofnon-recombinant MBL and one or more MASPs; (ii) incubating the complexin a suitable buffer to dissociate the MBL from the one or more MASPs;and (iii) separating the MBL from the one or more MASPs.
 5. Acomposition according to claim 4, wherein the buffer in step (ii) is anEDTA/acetate buffer at a pH of from 4.0 to 5.0.
 6. A compositionaccording to claim 4 or claim 5, wherein the buffer in step (ii)comprises NaCl.
 7. A composition according to any one of claims 4 to 6,wherein step (iii) includes a chromatographic method and/or filtration.8. A composition of claim 7, wherein the chromatographic method isselected from the group consisting of: size exclusion chromatography andion exchange chromatography.
 9. A method of producing a pharmaceuticalcomposition, the method comprising: (i) providing a complex ofnon-recombinant MBL and one or more MASPs; (ii) dissociating the MBLfrom at least some of the one or more MASPs; (iii) separating the MBLfrom at least some of the one or more MASPs; and (iv) admixing theresulting MBL from step (iii) with a pharmaceutically acceptable carrieror diluent.
 10. A method of claim 9, wherein step (ii) involvesincubating the complex in a suitable buffer.
 11. A method according toclaim 10, wherein the buffer is an EDTA/acetate buffer at a pH of from4.0 to 5.0.
 12. A method according to claim 10 or claim 11, wherein thebuffer comprises NaCl.
 13. A method according to any one of claims 9 to12, wherein step (iii) includes a chromatographic method and/orfiltration.
 14. A method of claim 13, wherein the chromatographic methodis selected from the group consisting of: size exclusion chromatographyand ion exchange chromatography.
 15. A method according to any one ofclaims 9 to 14, wherein step (i) comprises providing a side fractionfrom plasma fraction processes.
 16. A method according to claim 15,wherein step (i) further comprises separating complexes ofnon-recombinant MBL and one or more MASPS from other plasma proteinspresent in the side fraction from plasma fraction processes by mannanaffinity chromatography.
 17. A pharmaceutical composition obtained bythe method of any one of claims 9 to
 16. 18. The pharmaceuticalcomposition of claim 17, wherein the composition is substantially freeof activated MASPs.
 19. A method of treating or preventing a disease ina subject, the method comprising administering to the subject aneffective amount of a pharmaceutical composition according to any one ofclaims 1 to 8, 17 or
 18. 20. A method according to claim 19, wherein thesubject is a bone marrow allograft recipient.
 21. A method according toclaim 19, wherein the subject is immunodeficient.
 22. A method accordingto claim 19, wherein the subject has community acquired or nosocomialsepticaemia.
 23. A method according to claim 19, wherein the subject isa low birthweight and/or premature infant.
 24. A method according toclaim 19, wherein the subject is infected with a pathogen.
 25. A methodaccording to any one of claims 19 to 24, wherein the subject has an MBLdeficiency.
 26. A method according to claim 25, wherein the subject isan infant at risk from developing acute lymphoblastic leukaemia.
 27. Acomposition comprising isolated non-recombinant MBL, said compositionbeing substantially free of activated MASPs, for use prophylactically orin therapy.
 28. A composition comprising isolated non-recombinant MBL,said composition being substantially free of MASPs, for useprophylactically or in therapy.
 29. Use of a composition comprisingisolated non-recombinant MBL, said composition being substantially freeof MASPs, in the manufacture of a medicament for use in administering toa subject in need of said composition.
 30. Use according to claim 29,wherein the subject is a bone marrow allograft recipient.
 31. Useaccording to claim 29, wherein the subject is immunodeficient.
 32. Useaccording to claim 29, wherein the subject has community acquired ornosocomial septicaemia.
 33. Use according to claim 29, wherein thesubject is an infant at risk from developing has acute lymphoblasticleukaemia.
 34. Use according to claim 29, wherein the subject is a lowbirthweight and/or premature infant.
 35. Use according to claim 29,wherein the subject is infected with a pathogen
 36. Use according to anyone of claims 29 to 35, wherein the composition is substantially free ofMASPs.
 37. A peptide of formula X-R1-Arg-R2-Y, wherein R1-Arg-R2 is apeptide consisting of 6 or more contiguous amino acids derived from theMASP cleavage site of a complement protein; X is NH₂, a blocking groupor a detectable label; and Y is COOH or a detectable label, providedthat when X is NH₂ or a blocking group, Y is not COOH and when Y isCOOH, X is not NH₂ or a blocking group.
 38. A peptide according to claim37, wherein the complement protein is C4.
 39. A peptide according toclaim 38, wherein the C4 protein is human C4 and the cleavage sitecomprises Arg756.
 40. A peptide according to any one of claims 37 to 39,wherein X is a quencher molecule and Y is a fluorescent label, orvice-versa, such that a fluorescent signal is obtained when thesubstrate is cleaved.
 41. Use of a peptide according to any one ofclaims 37 to 40, in a method of determining the presence of MASPactivity in a sample.
 42. Use according to claim 41, wherein the sampleis a composition according to any one of claims 1 to 8, 17 or
 18. 43. Amethod of determining the presence of MASP activity in a sample whichmethod comprises contacting the sample with a peptide according to anyone of claims 37 to 40 and determining whether said peptide has beencleaved.
 44. A method according to claim 43, wherein the sample is acomposition according to any one of claims 1 to 8, 17 or
 18. 45. Amethod of producing a pharmaceutical composition according to any one ofclaims 1 to 3 which method comprises: (i) providing a complex ofnon-recombinant MBL and one or more MASPs; (ii) incubating the complexin a suitable buffer to dissociate the MBL from the one or more MASPs;(iii) separating the MBL from the one or more MASPs; (iv) screening theMBL obtained from (iii) for MASP activity using a method according toclaim 43; and (v) admixing the resulting purified MBL with apharmaceutically acceptable carrier or diluent.